The distribution of subtypes of the muscarinic receptor in homogenates of the rat brain was investigated by measuring the competitive inhibition of the binding [3H]N-methylscopolamine by pirenzepine and AF-DX 116 (11[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one). In most brain regions, the competitive binding curves for AF-DX 116 and pirenzepine were consistent with a two-site model. The dissociation constant of pirenzepine for its high-affinity site (M1 receptor) was approximately 10(-8) M, whereas the dissociation constant of AF-DX 116 for its high affinity site (M2 receptor) was approximately 10(-7) M. In many regions, particularly those in the forebrain, the sum of the densities of the M1 and M2 binding sites was substantially less than 100% of the total sites, indicating the existence of a third population of sites lacking high affinity for both pirenzepine and AF-DX 116. We have designated these latter sites as non-M1, non-M2 muscarinic receptors. In general, the densities of the M1 and non-M1, non-M2 binding sites were highest in cerebral cortex, corpus striatum and hippocampus, intermediate in thalamus and hypothalamus, and lowest in midbrain, medulla-pons and cerebellum, whereas the M2 binding site had a relatively low, uniform density throughout the brain. The binding capacity of [3H]N-methylquinuclidinyl benzilate was estimated to be 20 to 30% lower than that of [3H]quinuclidinyl benzilate in various regions of the forebrain, but not in more caudal regions of the brain where the two radioligands had approximately the same binding capacities. Treatment of homogenates of the cerebral cortex with benzilylcholine mustard caused a selective loss of the majority of the [3H]N-methylscopolamine binding sites but spared 25% of the sites labeled by [3H]quinuclidinyl benzilate The results of pirenzepine/[3H]quinuclinyl benzilate competitive binding experiments on cerebral cortex treated with benzilylcholine mustard showed that the residual binding sites for [3H] quinuclidinyl benzilate were enriched in M1 muscarinic receptors.